1. APOPTOSIS

2. APOPTOSIS

3. Normal development e.g. immune system
WHEN DOES APOPTOSIS OCCUR?
Normal development e.g. immune system

4. Disease states e.g. Alzheimer’s disease
WHEN DOES APOPTOSIS OCCUR?
Disease states e.g. Alzheimer’s disease
Amyloid plaques in the brain

5. TYPES OF CELL DEATH
NECROSIS
APOPTOSIS

6. Morphological NECROSIS loss of membrane integrity
TYPES OF CELL DEATH
Morphological
NECROSIS
loss of membrane integrity
swelling of cytoplasm & mitochondria
total cell lysis
no vesicle formation
disintegration of organelles
APOPTOSIS
Membrane blebbing, no loss of integrity
Aggregation of chromatin at nuclear membrane
Shrinking of cytoplasm & nuclear condensation
Fragmentation into smaller bodies
Formation of apoptotic bodies
Mitochondria become leaky

7. Biochemical NECROSIS APOPTOSIS Energy dependent
Loss of regulation of ion homeostasis
No energy requirement
Random digestion of DNA
Postlytic DNA fragmentaion
Tightly regulated process
APOPTOSIS
Energy dependent
Non random DNA fragmentation
Prelytic DNA fragmentation
Activation of caspase cascade
Release of various factors into cytoplasm

8. physiological NECROSIS APOPTOSIS Affects individual cells
Affects groups of cells
Evoked by non physiological disturbances
Phagocytosis by adjacent cells
Significant inflammatory response
APOPTOSIS
Affects individual cells
Induced by physiological stimuli
Phagocytosis by macrophages
No inflammatory response

9. Caspases – key executioners of apoptosis
(cysteinyl aspartate specific proteases)
Highly conserved proteases
14 homolgues
inactive zymogens
Caspases divided into
Initiator caspases: Caspases 2, 8,9,10 or
2) Effector caspases: caspase 3,6,

10. Properties of proteases
Irreversible -
Autocatalytic: triggered by cofactor binding or inhibitor removal
Proteases can regulate their own activation
Have protease, will have inhibitor
specificity

11. Caspase structure 3 domains 1) highly variable NH2 domain
2) large subunit (~20kD)
3) small subunit
( ~10kD)
Highly specific
absolute requirement for cleavage after aspartic acid
recognition of at least 4 amino acids NH2 terminals to the cleavage site

12. Caspase structure 2 key features:
variable N domain regulates activation
all domains derived from proenzyme at cleavage specific sites

13. Basic apoptotic machinery
DNA fragmentation,
chromatin condensation,
membrane blebbing,
cell shrinkage &
disassembly into apoptotic bodies
engulfment min
effectors responsible for cellular changes associated with apoptosis.
Caspases inactivate proteins that protect cells from apoptosis

14. How do caspases disassemble a cell? It slices, it dices
Selective cleavage of specific proteins
eg bcl-2, or CAD/ICAD
e.g. nuclear lamina
eg. Gelsolin

15. What triggers apoptosis?
Loss of contact with surroundings
Irreparable internal damage
Conflicting signals for cell division
Specific ‘death ligands’

16. How are caspases activated?
More than one way to skin a cat
Proteolytic cleavage
activation of downstream, effector caspases

17. How are caspases activated?
More than one way to skin a cat
Induced proximity
aggregation of multiple procaspase-8 molecules into close proximity somehow results in cross-activation

18. How are caspases activated?
More than one way to skin a cat
Holoenzyme formation
Activation of caspase-9 is mediated by means of conformational change, not proteolysis

19. The roads to ruin
The nematode C.elegans

20. The roads to ruin
Mammals

21. External signals driven by death receptors (DR) e.g. CD95 (or Fas/Apo)
Each CD95L trimer binds to 3 CD95 leading to DD clustering.
FADD ( Fas associated death domain/ Mort 1) binds via its own DD
Caspase –8 oligomerisation drives activation through self cleavage
Caspase –8 then activates downstream effector caspases like caspase –9 (CED-9 homolog)
Apoptosis initiation

22. Internal signals

23. BCL-2

24. apoptosis TRIGGER REGULATOR EXECUTIONER DNA damage Death receptors
Growth factor withdrawal
P53
Bcl-2 family
Death domain factor
Cytochrome c
oncogenes
Apaf-1
Caspases